Study On Degradation Mechanism And Purification Technique Of Lithospermic Acid B

Danshen, a traditional Chinese medicine, is the dried roots of Salvia miltiorrhiza Bge that belongs to the family of Labiatae. According to Chinese Pharmacopoeia 2005, Danshen has been judged to be highly effective in "opening blockage and relieving pain, activating circulation and dispersing stasis, clearing heat and tranquilizing mind". Lithospermic acid B (LAB), a polyphenolic acid, is the most common and abundant compound in danshen. Recent studies indicated that LAB has strong antioxidant ability and free radical scavenging activity. However, LAB is unstable in aqueous solution. Degradation of LAB resulted in the loss of the clinical efficacy and addition of unknown components in danshen products. Degradation problem has become a bottleneck in the pharmaceutical field. In this study, the kinetics and thermodynamics and advanced analysis methods, e.g. high performance liquid chromatography (HPLC), liquid chromatography-mass Spectrometry (LC-MS) and nuclear magnetic resonance (NMR), were employed to investigate the characters of kinetics and mechanism of degradation of LAB in aqueous solution. Based on this knowledge, a novel technique was developed for extraction and purification of LAB from danshen.Firstly, the degradation behaviors of phenolic acids extracted from danshen were investigated by HPLC on basis of 11 known phenolic acids identified by LC-MS. The results indicate that danshensu, protocatechuic aldehyde and caffeic acid increase as polyphenolic acids of LAB, salvianolic acid A and salvianolic acid E decline. Additionally, lithospermic acid and its isomers firstly increase and then decrease, indicating that they are prominent intermediates. Moreover, the hydrolysis depends significantly on pH of aqueous solution. The optimum pH is at pH 2-3 for stability of the similar polyphenolic acids due to their similar structures.Secondly, the degradation of LAB was investigated as a function of buffer concentration, pH and temperature. The degradation behavior follows pseudo-first-order kinetics under all experimental conditions. The results indicate that the observed rate constants increase linearly with the increase of buffer concentration. The pH-rate profile shows a characteristic S-shape, and the maximum stability was obtained at pH 2.0. At pH <2.0, the rate of degradation increased with decrease of pH value, indicating specific hydrogen-ion catalysis. In the pH range from 2.0 to 7.0, the degradation rate increased with increase of pH value, indicating specific hydroxyl-ion catalysis. The degradation rate increased with the increase of temperature. From experimental data, activation energies are calculated to be 40-85kJ/mol at pH 2.0-4.0. This implies that ester hydrolysis is a primary reaction in degradation.Thirdly, the chemical fate of LAB in aqueous solution was studied, based on separation and structure identification of five degradation products by ¹H (¹³C) NMR analysis, including danshensu, protocatechuic aldehyde, caffeic acid, lithospermic acid and salvianolic acid E. The primary degradation pathway is proposed to involve the cleavage of ester bond and ring-opened benzofuran in the LAB. Among degradation products, danshensu and lithospermic acid are produced through ester bonds hydrolysis, protocatechuic aldehyde through oxidation, and salvianolic acid E through ring-opened benzofuran in the LAB.Based on the kinetics and degradation mechanism of LAB, a novel technique for extraction and purification of LAB was established, including microwave-drying fresh danshen, aqueous two-phase extraction, and purification of LAB by polyamide adsorption.To begin with, a novel method combined microwave-drying fresh danshen and maceration at room temperature was used to extract LAB from danshen. The results show that the best quality of product was observed in the drying period with 650W. The dissolution rate of LAB was 2.17 times higher than the sun-drying sample, and obtain higher yield of LAB in maceration process.What's more, an aqueous two-phase system comprised of ethanol and (NH₄)₂SO₄ was evaluated to separate LAB from crude extract using partition coefficient and recovery as target indicator. The results show that the partition coefficient and the yield of LAB increase with the mass fractions of (NH₄)₂SO₄ and ethanol, but they increase at first and then decrease when the pH value increases. The partition coefficient of LAB reaches to 95.58 and its recovery to 97.73 % at pH 2.0 in an aqueous two-phase system composed of 20 wt % ethanol and 25 wt % (NH₄)₂SO₄. Thus aqueous two-phase system may provide a new technique for extraction of LAB from the crude extract.In addition, the adsorption of LAB onto polyamide was studied. The different parameters affecting the adsorption capacity were investigated, such as initial LAB concentration, adsorption time, pH of solution and temperature. The experimental data were exploited for kinetics and thermodynamic evaluation related to the adsorption process. The results showed the adsorption isotherms correlated well with the Langmuir type adsorption isotherm, and adsorption capacity were found to be 69, 196, 376mg/g at initial concentration of LAB of 0.16, 0.43, 2.66mg/mL at 25℃, respectively. Adsorption process follows pseudo-second order type adsorption kinetics. The pH of the aqueous solution is one of important controlling parameters in the adsorption process. The optimum pH range for higher capacity of LAB is at pH <3.0. At pH3.0-5.0, the adsorption capacity of LAB rapidly decreases with increase of pH value. At pH>5.0, a lower adsorption capacity of LAB was observed. This behavior implies that adsorption of LAB onto polyamide from aqueous solution is based on hydrogen bond and electrostatic interaction. Thermodynamic parameters such as standard free energy (ΔG⁰ ), standard enthalpy (ΔH⁰), and standard entropy (ΔS⁰) were calculated from experimental data. The negative value ofΔH⁰ (-50.0kJ/mol) shows that the adsorption is exothermic process, and the negative value ofΔS⁰ (-147.3J/mol) indicates a greater order of reaction during the adsorption. The values ofΔG⁰ were found to be -6.17, -5.40, -4.64 and -3.22kJ/mol at 25, 30, 35, 45℃, respectively. The negative values ofΔG⁰ indicate that adsorption of LAB onto polyamide is a spontaneous process. The values of the standard free energy (ΔG⁰ ) decrease with the increase of the temperature, so the adsorption is favored at lower temperature.Finally, a rapid method to purify LAB was established using polyamide column chromatography. Many factors were studied, including polyamide particle-size, pH of solution, flow rate and concentration of LAB in feed solution. If polyamide particle-size is 60-100 mesh, the optimum parameters are as follows: 2.4 mg/mL of concentration of LAB in crude extract, pH 2.0 of solution and 1.0 mL/min of flow rate. After the extract was loaded onto the column, polyamide bed was eluted by 3BV (three folds volume of resin bed) water to wash impurity from the crude extract, and the column was then eluted sequentially by 1BV 20% and 5BV 70% of ethanol, respectively. The fraction at 70% ethanol was collected and evaporated. The concentrate was freeze-dried and the sample containing 53.53% LAB was obtained. The recovery is 83.00% and the yield to danshen roots is 2.29%. If polyamide particle-size was 100-200 mesh , and the flow rate was 0.5mL/min, the polyamide bed was eluted by 3BV water to remove impurity, and then the column was eluted sequentially by 1BV 20%,1.5BV 50%, and 1.5-4BV 70% of ethanol, respectively. The fraction at 70% ethanol containing LAB was collected and evaporated. The condensate was freeze-dried and the sample containing over 95% LAB was obtained, the recovery is only 30%.In conclusion, the hydrolysis of ester bond and ring-opened benzofuran in the LAB are the primary degradation pathways, and temperature and pH of the aqueous solution are two important controlling parameters. A novel method combined microwave-drying fresh danshen and maceration significantly increases the dissolution rate of LAB from danshen. Aqueous two-phase extraction in combination with polyamide column chromatography can purify LAB from crude extract. The present study might provide scientific information for LAB in pharmaceutical field.